Cryo-EM grid preparation and data collection

For the preparation of cryo-EM grids, 2.6 μL of purified complexes at ~10 mg/mL for the TA–PAR1–G_q complex and ~5.0 mg/mL for the TA–PAR1–G_i complex were applied to glow-discharged holey carbon grids (Quantifoil R1.2/1.3), respectively. The grids were vitrified in liquid ethane by Vitrobot Mark IV (FEI). Frozen grids were transferred to liquid nitrogen and stored until data collection. Cryo-EM imaging was performed on a Titan Krios (FEI) at 300 kV using a Gatan K3 summit direct electron detector with a Gatan energy filter with a slit width of 20 eV at the Center of Cryo-Electron Microscopy Research Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences (Shanghai, China). Micrographs were recorded by SerialEM software in super-resolution mode at a magnified physical pixel size of 1.071 Å, with defocus values ranging from −0.5 μm to −3.0 μm. The total exposure time was set to 3 s with intermediate frames recorded every 0.083 s, resulting in an accumulated dose of ~70 electrons per Ų and a total of 36 frames per movie stack. A total of 5048 and 6097 movies were collected for the TA–PAR1–G_q complex and the TA–PAR1–G_i complex, respectively.